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- PDB-6smh: Cryo-electron microscopy structure of a RbcL-Raf1 supercomplex fr... -

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Basic information

Entry
Database: PDB / ID: 6smh
TitleCryo-electron microscopy structure of a RbcL-Raf1 supercomplex from Synechococcus elongatus PCC 7942
Components
  • Ribulose bisphosphate carboxylase large chain
  • Rubisco accumulation factor 1 (RAF1) peptide
KeywordsPHOTOSYNTHESIS / Rubisco / Rubisco accumulation factor1 / Raf1 / Synechococcus elongatus 7942 / Cyanobacteria
Function / homology
Function and homology information


ribulose bisphosphate carboxylase complex assembly / carboxysome / photorespiration / ribulose-bisphosphate carboxylase / carbon fixation / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / photosynthesis / monooxygenase activity / magnesium ion binding / cytoplasm
Similarity search - Function
Rubisco accumulation factor 1 / Rubisco accumulation factor 1, helix turn helix domain / Rubisco accumulation factor 1, C-terminal / Rubisco accumulation factor 1, alpha helical domain / Rubisco Assembly chaperone C-terminal domain / Rubisco accumulation factor 1 alpha helical domain / Rubisco accumulation factor 1 helix turn helix domain / Ribulose bisphosphate carboxylase large subunit, type I / Ribulose bisphosphate carboxylase, large chain, active site / Ribulose bisphosphate carboxylase large chain active site. ...Rubisco accumulation factor 1 / Rubisco accumulation factor 1, helix turn helix domain / Rubisco accumulation factor 1, C-terminal / Rubisco accumulation factor 1, alpha helical domain / Rubisco Assembly chaperone C-terminal domain / Rubisco accumulation factor 1 alpha helical domain / Rubisco accumulation factor 1 helix turn helix domain / Ribulose bisphosphate carboxylase large subunit, type I / Ribulose bisphosphate carboxylase, large chain, active site / Ribulose bisphosphate carboxylase large chain active site. / Ribulose bisphosphate carboxylase, large subunit, ferrodoxin-like N-terminal / Ribulose bisphosphate carboxylase large chain, N-terminal domain / Ribulose bisphosphate carboxylase, large subunit, C-terminal / RuBisCO / Ribulose bisphosphate carboxylase, large subunit, C-terminal domain superfamily / RuBisCO large subunit, N-terminal domain superfamily / Ribulose bisphosphate carboxylase large chain, catalytic domain
Similarity search - Domain/homology
Ribulose bisphosphate carboxylase large chain / RuBisCO accumulation factor 1
Similarity search - Component
Biological speciesSynechococcus elongatus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsHuang, F. / Kong, W.-W. / Sun, Y. / Chen, T. / Dykes, G.F. / Jiang, Y.L. / Liu, L.N.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Royal SocietyURF/R/180030 United Kingdom
Royal SocietyUF120411 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M024202/1 United Kingdom
Leverhulme TrustECF-2016-778 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Rubisco accumulation factor 1 (Raf1) plays essential roles in mediating Rubisco assembly and carboxysome biogenesis.
Authors: Fang Huang / Wen-Wen Kong / Yaqi Sun / Taiyu Chen / Gregory F Dykes / Yong-Liang Jiang / Lu-Ning Liu /
Abstract: Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes ...Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer-dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO-fixing modules to improve photosynthesis.
History
DepositionAug 21, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 8, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed ..._citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Aug 5, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: Ribulose bisphosphate carboxylase large chain
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose bisphosphate carboxylase large chain
E: Ribulose bisphosphate carboxylase large chain
F: Ribulose bisphosphate carboxylase large chain
G: Ribulose bisphosphate carboxylase large chain
H: Ribulose bisphosphate carboxylase large chain
I: Rubisco accumulation factor 1 (RAF1) peptide
J: Rubisco accumulation factor 1 (RAF1) peptide
K: Rubisco accumulation factor 1 (RAF1) peptide
L: Rubisco accumulation factor 1 (RAF1) peptide
M: Rubisco accumulation factor 1 (RAF1) peptide
N: Rubisco accumulation factor 1 (RAF1) peptide
O: Rubisco accumulation factor 1 (RAF1) peptide
P: Rubisco accumulation factor 1 (RAF1) peptide


Theoretical massNumber of molelcules
Total (without water)570,54216
Polymers570,54216
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area66470 Å2
ΔGint-141 kcal/mol
Surface area196660 Å2
MethodPISA

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Components

#1: Protein
Ribulose bisphosphate carboxylase large chain / RuBisCO large subunit


Mass: 49641.219 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (strain PCC 7942 / FACHB-805) (bacteria)
Gene: cbbL, rbcL, Synpcc7942_1426 / Production host: Escherichia coli (E. coli)
References: UniProt: Q31NB3, ribulose-bisphosphate carboxylase
#2: Protein
Rubisco accumulation factor 1 (RAF1) peptide


Mass: 21676.561 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (strain PCC 7942 / FACHB-805) (bacteria)
Gene: Synpcc7942_0833 / Production host: Escherichia coli (E. coli) / References: UniProt: Q31Q05

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RbcL-Raf1 supercomplex of Synechococcus elongatus 7942
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Synechococcus elongatus (strain PCC 7942 / FACHB-805) (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 25 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16022 / Symmetry type: POINT

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